Date of Award

Fall 2009

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Stacia A Sower

Abstract

Hagfish and lampreys are the only two representatives of Agnathans among extant vertebrates. The regulatory hypothalamic neurohormone, gonadotropin-releasing hormone (GnRH), regulates reproduction in all vertebrates through the hypothalamic-pituitary-gonadal axis. Most vertebrates have at least two forms of GnRH in the brain. In hagfish the primary amino acid structure of GnRH has not been identified as yet, however, indirect methods have shown an immunoreactive GnRH or a GnRH-like peptide in the brain of hagfish. In addition concentrations of brain immunoreactive-GnRH have been correlated with reproductive stages in the Atlantic hagfish ( Myxine glutinosa). Therefore the objective of the first study was to identify the primary structure of putative GnRH(s) by using PCR, MALDI, and chromatography (column and HPLC). However, a GnRH-like molecule was not identified despite extensive efforts and experiments.

My second objective was to identify a novel GnRH from lamprey brain using in silico analysis. A cDNA encoding a novel GnRH, named lamprey GnRH-II, was cloned from the sea lamprey. The deduced amino acid sequence of the newly identified lamprey GnRH-II is QHWSHGWFPG. The architecture of the precursor is similar to that reported for other GnRH precursors consisting of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide, however, the gene for lamprey GnRH-II does not have introns in comparison with the gene organization for all other vertebrate GnRHs. Lamprey GnRH-II precursor transcript was expressed in a variety of tissues. In situ hybridization of the brain showed expression and localization of the transcript in the hypothalamus, medulla, and olfactory regions, whereas immunohistochemistry using a specific antiserum showed only lamprey GnRH-II cell bodies and processes in the preoptic nucleus/hypothalamus areas. Lamprey GnRH-II was shown to stimulate the hypothalamic-pituitary axis using in vivo and in vitro studies. Lamprey GnRH-II was also shown to activate the inositol phosphate signaling system in COS-7 cells transiently transfected with the lamprey GnRH receptor. These studies provide evidence for a novel lamprey GnRH that has a role as a third hypothalamic GnRH. The newly discovered lamprey GnRH-II offers a new paradigm of the origin of the vertebrate GnRH family.

Download

Share

COinS